The human-specific bacterial pathogen Neisseria gonorrhoeae (Ngo) causes the sexually-transmitted infection gonorrhea. Ngo expresses outer membrane transporters to acquire iron and other metals in a TonB-dependent manner. In order to prevent microbial growth and infectivity, humans use high affinity metal-binding proteins to sequester essential metals, a phenomenon known as ?nutritional immunity.? Ngo subverts nutritional immunity by using dedicated transporters that bind to and remove the metals from human metal-binding proteins such as transferrin and lactoferrin. Our preliminary data indicates that two poorly-characterized Ngo outer membrane transporters, TdfH and TdfJ, enable Ngo to internalize zinc (Zn). Furthermore, we show that TdfH affords Ngo the ability to overcome Zn sequestration imposed by the innate immunity protein, calprotectin (CP), which is produced in abundance by neutrophils and macrophages and accumulates to high concentrations at sites of inflammation. We hypothesize that TdfH and TdfJ are Zn transporters that enable Ngo to overcome the growth inhibitory effects of the innate immunity proteins produced during inflammation. These studies are significant because knowing how Ngo acquires essential metals like Zn from the human host opens new opportunities for developing new therapeutics against `superbug' strains, whether they be targets for a protective vaccine or small molecule inhibitors of crucial transporters. The overarching hypothesis to be tested in the proposed study is that Ngo subverts nutritional immunity imposed by S100 proteins produced by neutrophils and macrophages by the deployment of outer-membrane transporters that bind to and relieve these proteins of their sequestered transition metal cargo. The specific aims are as follows: Aim 1 will define the characteristics of ligand interactions with TdfH and TdfJ using crystallography, biophysical approaches to protein-protein interactions and mutagenesis to confirm interactions. We will also ascertain if ligand interactions are specific to human proteins, given that humans are the only natural host for Ngo. Aim 2 will define the nutritional and metal environment sensed by Ngo upon exposure to human immune cells and secretions, employing mass spectrometry and imaging techniques alongside bacterial gene expression. Aim 3 will explore how Zn uptake systems impact survival of Ngo after exposure to human immune cells and secretions that contain S100 proteins. These studies will utilize Ngo survival assays with macrophages and neutrophils, combined with real-time confocal visualization and S100 inhibition studies. Overall these innovative studies will provide an in-depth analysis of two new outer membrane transport proteins involved in Zn acquisition and will provide novel insights into the human environmental niches in which Ngo normally resides.